Substrate determining method

ABSTRACT

There is provided a method for precisely quantitating a substrate by means of a measurement system with a simple structure without occurrence of a measurement error due to an interfering substance. In a method for quantitating a substrate in a sample solution which contains a dissolved interfering substance and the substrate, by the use of an electrode system and a reagent system, (a) a sample solution which contains a dissolved interfering substance and a substrate is supplied to an electrode system comprising a working electrode and a counter electrode under the existence of a reagent system comprising oxidoreductase and an electron mediator; (b) an AC potential is applied to the working electrode, to cause a redox reaction of the electron mediator: (c) an electric signal produced on the basis of the redox reaction is measured by means of the electrode system; and (d) the substrate is quantitated on the basis of the electric signal.

TECHNICAL FIELD

The present invention relates to a method for electrochemicallyquantitating a substrate contained in a sample with the use ofoxidoreductase.

BACKGROUND ART

There have been developed plenty of methods for simple measurement andquantitation of a specific substrate present in a sample. In particular,methods for measurement and quantitation with high selectivity byutilizing a substrate-selective catalytic action of an enzyme hasrecently been drawing much attention, and some of these methods havebeen used as methods for quantitating a specific element in body fluidin the field of clinical examinations, and further in the field ofself-examinations by ordinary people.

As an example of methods for quantitating and measuring a substrate in asample, an electrochemical quantitation method of glucose is described.Glucose oxidase (hereinafter abbreviated to GOx) is an enzyme thatselectively catalyzes oxidation of glucose. When a certain amount of anoxidized form of electron mediator (compound for transferring electrons,generated due to a reaction, from an enzyme to an electrode) is madepresent in a reaction solution containing GOx and glucose, glucoseoxidation leads to reduction of the oxidized form of electron mediator,to produce a reduced form of electron mediator. The produced reducedform of electron mediator is oxidized by the use of an electrode with aDC potential applied thereto so as to measure a flowing current. Glucosecan be quantitated by such a measurement because the flowing current inthis case is proportional to an amount of the reduced form of electronmediator produced due to the reaction of GOx with glucose, and theamount of the reduced form of electron mediator is proportional to theglucose content.

Further, a biosensor electrode device can be produced by getting boththe enzyme and electron mediator dry on the electrode to be carried. Thedevelopment of disposable-type glucose sensors based on such technologyhas recently been attracting a great deal of attention. Onerepresentative example is a biosensor described in the specification ofJapanese Patent No. 2517153. A disposable-type glucose sensorfacilitates measurement of the glucose concentration by simpleintroduction of a sample solution into a sensor device detachablyconnected to a measurement device.

While the method for simple measurement and quantitation of a specificsubstrate present in a sample was described in the above, other than themethod for quantitating a substrate, there exists a method as one ofgeneral electrochemical measurements, which comprises application of nota DC potential but an AC potential to an electrode, and measurement ofan obtained electric signal (hereinafter referred to as an alternatingcurrent method). This is a method for primarily obtaining information ona structure of an interface between an electrode and electrolyte, andfor example, characteristic evaluations of electrodes carrying activematerials in secondary batteries have been conducted using such ameasurement method.

Although, as thus described, the use of an enzyme can result inrealization of measurement with relatively high selectivity of asubstrate, a measurement error has been induced by the influence ofother substances than a substrate as a subject to be measured which iscontained in a sample in the conventionally-used electrochemicalmeasurement where a DC potential is applied. For example, when anelectrochemical measurement is conducted using blood as a sample,easily-oxidizable compounds contained in the blood, such as ascorbicacid (vitamin C), uric acid and acetaminophen, may bring about an error.Such a substance as induces an error in a measurement where the DCpotential is applied is called an interfering substance.

In the following, the reason for occurrence of a current error due to aninterfering substance in the conventional method is described, usingFIG. 5. FIG. 5 is a graph showing an example of the relationship betweenthe electrode potential and current, obtained in a solution where GOx,an electron mediator and glucose are dissolved, and a solution of aninterfering substance. In the glucose quantitation by the conventionalmethod, application of a potential E1, which allows sufficient oxidationof an electron mediator, to an electrode usually generates a current.When E1 is applied to the electrode, electrochemical oxidation of aninterfering substance at the electrode proceeds sufficiently, as evidentfrom FIG. 5. Since superimposition of a current (I3) that flows due tothe electrochemical oxidation of the interfering substance on a current(I1) due to glucose causes an error in the glucose measurement. Grantingthat the electron mediator uses a potential to be moderately oxidized,e.g. E2 shown in FIG. 5, a current due to the interfering substancecannot be prevented, and further, a proportion of the current due to theinterfering substance in the entire current increases to deteriorate anS/N ratio.

When a compound having redox potential, which is more negative than apotential E3 with which an interfering substance is not oxidized, isused as an electron mediator, there should theoretically be nooccurrence of an error. Several attempts have been made to conduct theglucose quantitation using such a compound. In this case, however, apotential difference between GOx and the compound becomes smaller,thereby considerably slowing the transfer rate of electronstherebetween, or preventing the electrons from transferring at all. As aresult, problems may arise that a current for the glucose quantitationdoes not become large enough to be detectable, or it takes extremelylong period of time to detect the current, and further the current isnot obtained at all.

Moreover, the concentration of an interfering substance in blood differsamong individuals, or even in blood of one individual, it differs everyday. It is therefore very difficult in the conventional measurement topredict a measurement error that may occur in measuring the interferingsubstance concentration in blood and then correct it.

A variety of measures have been attempted to correct or remove theinfluence of an interfering substance. The U.S. Pat. No. 6,340,428publication for example discloses a method as well as a sensor, in whichthe influence of an interfering substance is corrected by placement of athird electrode for measurement of an interfering substance in additionto a working electrode and a counter electrode. In advance of progressof an enzyme reaction and a subsequent measurement of a substrate at aworking electrode, an interfering substance contained in a sample ismeasured at a third electrode, whereby a favorable correction has beenrealized.

As a method for removing the influence of an interfering substancedeveloped has been a method of suppression of a current due to aninterfering substance by forming, on an electrode, a film for blockingdiffusion of the interfering substance to the electrode. For example,Wang. J et al. has disclosed the use of a poly (o-phenylene diamine)film in “Electroanalysis”, August, 1996, Pages 1127-1130.

As thus described, it has been necessary to complexify a structure of asensor device or an electrode in order to realize correction or removalof the influence of an interfering substance in an electrochemicalmethod using the conventional DC potential, which is used formeasurement and quantitation of a specific substrate present in asample.

In view of the aforesaid drawbacks in the prior art, it is an object ofthe present invention to provide a method enabling a precisequantitation of a substrate contained in a sample solution by means of ameasurement system with a simple structure, without causing ameasurement error due to an interfering substance.

DISCLOSURE OF INVENTION

The present invention relates to a method for quantitating a substratein a sample solution, which contains a dissolved interfering substanceand the substrate, by the use of an electrode system and a reagentsystem, comprising the steps of: (a) supplying a sample solution whichcontains a dissolved interfering substance and a substrate to anelectrode system comprising a working electrode and a counter electrodeunder the existence of a reagent system comprising oxidoreductase and anelectron mediator; (b) applying an AC potential to the workingelectrode, to cause a redox reaction of the electron mediator; (c)measuring an electric signal produced on the basis of the redoxreaction, by means of the electrode system; and (d) quantitating thesubstrate on the basis of the electric signal.

It is preferable that in the step (a), the working electrode and counterelectrode are disposed on the same plane.

It is also preferable that in the step (a), the working electrode andcounter electrode are disposed in positions opposed to each other acrossa space.

It is preferable that the method for quantitating a substrate furthercomprises a step (e) of applying a DC potential to the workingelectrode, and a step (f) of measuring an electric signal produced inthe step (e).

It is preferable that in the step (b), a central potential of the ACpotential is within the range of −0.1 to +0.1 V relative to a redoxpotential of the electron mediator, and is a potential more positivethan a potential that is 0.05 V negative relative to the most negativepotential in a potential region where a reaction of the interferingsubstance at the working electrode is diffusion-controlled.

It is also preferable that the electric signal is impedance.

It is also preferable that the electrode system further comprises areference electrode.

It is also preferable that the working electrode is a rotating discelectrode or a micro-electrode.

It is preferable that oxidoreductase is glucose oxidase orpyrroloquinoline quinone-dependent glucose dehydrogenase, and theelectron mediator is ferrocene carboxylic acid.

It is also preferable that oxidoreductase is pyloroquinolinequinone-dependent glucose dehydrogenase, and the electron mediator isruthenium hexacyanate.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an oblique view of a biosensor used in one example of thepresent invention, from which a reagent system has been removed.

FIG. 2 is a vertical sectional view (sectional view taken on the lineX-X) showing the main part of the biosensor shown in FIG. 1.

FIG. 3 is a graph in which real number elements and imaginary numberelements of impedance Z are plotted in an example of the presentinvention.

FIG. 4 is a diagram showing a construction of one example of measurementdevices for use in implementation of a quantitation method of asubstrate in accordance with the present invention.

FIG. 5 is a graph showing the relationship between the electrodepotential and current, which is obtained with a solution where GOx, anelectron mediator and glucose are dissolved and a solution of aninterfering substance.

BEST MODE FOR CARRYING OUT THE INVENTION

A method for quantitating a substrate in accordance with the presentinvention is characterized by comprising the steps of: (a) supplying asample solution which contains a dissolved interfering substance and asubstrate to an electrode system comprising a working electrode and acounter electrode under the existence of a reagent system comprisingoxidoreductase and an electron mediator; (b) applying an AC potential tothe working electrode, to cause a redox reaction of the electronmediator; (c) measuring an electric signal produced on the basis of theredox reaction, by means of the electrode system; and (d) quantitatingthe substrate on the basis of the electric signal.

According to such a method for quantitating a substrate in accordancewith the present invention, it is possible to measure a substratewithout the influence of an interfering substance. The reason for thisis described below.

In an alternating current measurement, an AC potential, which uses acertain DC potential as a central potential and contains, bysuperimposing, an AC potential element with very small amplitude versusthe central potential, is applied to a working electrode, and then anobtained electric signal is measured. As a measurement device used forexample is the one as shown in FIG. 4.

The measurement device shown in FIG. 4 comprises a waveform generator 2,an i/E converter 3, a rock-in-amplifier 4, a potentiostat 5, and alowpass filter 6. The waveform generator 2 generates an AC potential andthe potentiostat 5 controls a central potential. Subsequently, a currentobtained by a reaction at a working electrode in a biosensor 1 isconverted into a voltage signal with the i/E converter 3, and analternate current element and a direct current element are then obtainedwith the rock-in-amplifier 4 and the lowpass filter 6, respectively. Theuse of these elements enables quantitation of a substrate, as describedlater.

The alternating current measurement is described in the following, ascomparing with direct current cyclic voltammetry. First considered isdirect current cyclic voltammetry in the case of using a samplecontaining no interfering substance dissolved, and the same electrodesystem and reagent system as in the method for quantitating a substratein the above embodiment. In this case, so-called catalytic waves areobserved between a current (I) and a potential (E). Further, even in thecase of making a potential scanning rate faster, sigmoid waves having noanodic (or cathodic) peak derived from an electron mediator can usuallybe obtained. When the direct current cyclic voltammetry is performed insuch a system that the sigmoid waves can be obtained, by the use of arelatively slow potential scanning rate, almost no hysteresis isobserved between the anodic wave and cathodic wave. In this case,therefore, a rate (dI/dE) of current variations versus potentialvariations represents a reciprocal number of a resistance element (R) ofthe system, substantially in line with the Ohm's law.

Next considered is the case of conducting an alternating currentmeasurement in the same system. As in the aforesaid case of almost noexistence of hysteresis in the direct current cyclic voltammetry using arelatively slow potential scanning rate, almost no phase differenceoccurs between an alternating current (I_(ac)) and an AC potential(E_(ac)) in an alternating current measurement using a relatively lowfrequency. Hence impedance (Z) as a resistance element of the system inthis case is based on dI_(ac)/dE_(ac). Z is measured, and then the realnumber elements thereof are plotted as abscissa and the imaginary numberelements as ordinate (complex element impedance plotting). There beingalmost no phase difference between the current and potential at arelatively low frequency, as described above, a plot group exists in thevicinity of the real number axis, having a certain magnitude |Z_(o)|. Asthe frequency is gradually increased, the magnitude of Z decreases bythe influence of the capacity element of the system, and the plot groupthen forms a curved line that can be approximated by a circular arc witha diameter of |Z_(o)|.

dI_(ac)/dE_(ac) in the alternating current method increases withincreasing amount of a substrate contained in the system, as in the caseof the direct current cyclic voltammetry. Accordingly, |Z_(o)| in thecomplex impedance plot decreases with increasing substrate amount, andas a result, a diameter of an obtained circular arc decreases withincreasing substrate amount. It is therefore possible to measure orquantitate an amount of a substrate contained in a sample solution bymeasurement of an electric signal obtained by the alternating currentmethod. It is preferable here that a central potential of an ACpotential to be applied to a working electrode is set in the vicinity ofa redox potential of an electron mediator (e.g. in the vicinity of E2 inFIG. 5) because the setting causes increases in dI_(ac)/dE_(ac) and invariation amount of an electric signal accompanied by variations inamount of the substrate.

Meanwhile considered is the case of using an electrolyte singlycontaining an interfering substance. In this case, in a potential regionwhere the reaction of the interfering substance at an electrode isalmost diffusion-controlled (e.g. E4 to E1 in FIG. 5), dI/dE in directcurrent cyclic voltammetry becomes such a magnitude as is substantiallyalmost ignorable. In the alternating current measurement, therefore,when a central potential of an AC potential to be applied to a workingelectrode is in the potential range as described above, there issubstantially almost no current variation (dI_(ac)/dE_(ac)) derived fromthe interfering substance versus current modulation.

When a plurality of electrochemical reactions are simultaneouslyproceeding at an electrode, an obtained current is basically the simplesum of currents flowing due to the respective electrochemical reactions.In an electrolyte obtained by combining the two solution compositions inFIG. 5, for example, an obtained current with the potential E1 used is:I=I1+I3. As thus described, in arguments on the relationship between thecurrent and potential and also R and Z to be obtained from thatrelationship, even when a sample solution with a substrate and aninterfering substance mixed therein is used and the same electrodesystem and reagent system as in the aforesaid method for quantitating asubstrate in the present embodiment are used, both an explanation on theelectrolyte containing such a substrate as described above and anexplanation on an electrolyte singly containing an interfering substanceare established in respect to the substrate and interfering substance,respectively. As a result, for example, dI_(ac)/dE_(ac) in the vicinityof the potential E2 is substantially almost the same as that obtained inan electrolyte containing the aforesaid substrate while not containingan interfering substance, and hence the interfering substancesubstantially makes almost no contribution to dI_(ac)/dE_(ac).

For the reason described above, according to the method for quantitatinga substrate in one embodiment of the present invention, it is possibleto quantitate a substrate without the influence of an interferingsubstance.

Further, in the step (a) of the method for quantitating a substrate inaccordance with the present invention, disposition of the workingelectrode and counter electrode on the same plane is preferred. Thismakes a simpler substrate quantitation possible.

In this case used may be a biosensor comprising an insulating (first)base plate, an electrode system which comprises a working electrode anda counter electrode that are disposed on the aforesaid (first) baseplate, and a reagent system comprising a reagent system which containsoxidoreductase and an electron mediator.

It is also preferable that in the step (a), the working electrode andcounter electrode are disposed in positions opposed to each other acrossa space.

In this case used may be a biosensor comprising an insulating first baseplate, an insulating second base plate, an electrode system whichcomprises a working electrode disposed on the first base plate and acounter electrode disposed on the second base plate, and a reagentsystem which comprises oxidoreductase and an electron mediator.

It is preferable here that the method for quantitating a substrate inaccordance with the present invention further comprises a step (e) ofapplying a DC potential to the working electrode, and a step (f) ofmeasuring an electric signal produced in the step (e). In such a manner,the electric signal to be measured in the step (f) includes informationon the substrate and interfering substance. Accordingly, the combinationof this measurement with the alternating current measurement allowsquantitation of the interfering substance as well as the substrate,based on the electric signal measured in the step (c) and the electricsignal measured in the step (f).

It is also preferable that a central potential of the AC potential isset in the vicinity of a redox potential of the electron mediator. Inparticular, the central potential is preferably within the range of −0.4to +0.4 V, and more preferably within the range of −0.1 to +0.1 V,relative to the redox potential of the electron mediator.

It is further preferable that the central potential (E_(cen)(V)) of theAC potential is set within, or in the vicinity of, a potential regionwhere the reaction of the interfering substance at the working electrodeis diffusion-controlled. It is particularly preferable that the centralpotential is a potential more positive than a potential that is 0.05 Vnegative relative to the most negative potential (E_(min)(V)) in thepotential region where the reaction of the interfering substance at theworking electrode is diffusion-controlled. That is, the centralpotential and the most negative potential preferably satisfy:E_(cen)>E_(min)−5 (V). In this way, the current variation derived fromthe electron mediator versus modulation of the potential applied to theworking electrode can be made greater, while the current variationderived from the interfering substance can be made almost zero. Hence itis possible to completely remove the influence of the interferingsubstance in the substrate measurement by the alternating current methodusing the sample solution containing the substrate and interferingsubstance mixed.

It should be noted that the redox potential of the aforesaid electronmediator and the potential at which the reaction of the interferingsubstance is diffusion-controlled can be estimated by cyclicvoltammetry, wherein a 0.1 M phosphate buffer (pH=7.0) where theaforesaid substance is dissolved is used as an electrolyte, and a glassycarbon or metal electrode as a working electrode in combination with asuitable reference electrode are used.

Furthermore, the redox potential of the electron mediator carried by thebiosensor can be estimated as below. The electron mediator was dissolvedand extracted from the biosensor together with a coexisting substancewith the use of the aforesaid phosphate buffer to obtain a solution, andthe redox potential can then be estimated by cyclic voltammetry, usingthe obtained solution.

Herein, the AC potential to be applied to the working electrode is apotential (voltage) versus the counter electrode or the referenceelectrode which is used in combination with the working electrode.Further, an AC potential (voltage) in the present invention may be apotential having a waveform which successively or discretely modulatesdepending on time “t”, and is a concept including an approximate ACpotential. More specifically, the AC potential in the present inventionmay be a potential that satisfies, at least periodically:E(t1)<E(t2) and E(t2)>E(t3), orE(t1)>E(t2) and E(t2)<E(t3),where t1<t2<t3. The examples of such waveforms may include sine waves,square waves and step waves.

The electric signal to be used in the method for quantitating asubstrate in accordance with the present invention may be an electricsignal which varies with the progress of the electrochemical reaction,and may be exemplified by a current, admittance and impedance. Inparticular, when a potential having sine waves, or step waves which cansubstantially approximate sine waves, is applied, the electric signal ispreferably impedance. While a current and admittance may also be used asthe electric signals, it is preferable that these are converted intoimpedance and then the amount of the substrate is output based on theobtained impedance. In the case of applying a potential with squarewaves, a current is obtained as the electric signal and, based ondependency (variations) of the electric signal on (with) time and the ACpotential, information regarding the substrate amount can be obtained.

It is also preferable that the electrode system further comprises areference electrode. This arrangement stabilizes a potential to beapplied to the working electrode, thereby allowing a more stablesubstrate measurement. While the reference electrode to be used can bean Ag/AgCl electrode, a saturated calomel electrode (SCE) or the like,it is not limited to these and any electrode with a stable potential maybe used.

As for the working electrode to be used in the method for quantitating asubstrate in accordance with the present invention, aconventionally-known ones can be used without a specific limitation. Itis particularly preferable that the working electrode is a rotating discelectrode (hereinafter abbreviated to RDE) or a micro-electrode. The useof RDE which rotates at a fixed rate, or the use of a micro-electrodewhich has an electrode area of such a small size that a lateraldiffusion of the electron mediator toward the electrode surfacecontributes to the electrochemical reaction, allows larger currentvariation derived from the electron mediator versus modulation of thepotential applied to the working electrode, compared with the case ofusing a static bulk electrode. This can thus enhance the sensitivitytoward the substrate. The radius of the micro-electrode is preferablyfrom not longer than 50 μm (in the case of a circle electrode), and morepreferably from not longer than 50 am and not shorter than 20 μm. Thesubstrate measurement by means of the alternating current methodaccording to the present invention can be stably conducted by the use ofhigher frequency, as suggested, in direct current voltammogram usingthese electrodes, by the fact that hysteresis between the anodic waveand cathodic wave is very small even when no catalyst reaction occursand when a potential scanning rate is made relatively faster.

As for the sample solution, where an interfering substance is dissolved,to be used in the method for quantitating a substrate in accordance withthe present invention, a solution where a substrate and an interferingsubstance are dissolved, and further a living organism solution where asubstrate and an interfering substance are dissolved, e.g. blood,plasma, serum, urine and interstitial fluid, can be used. As forexamples of the interfering substance cited can be ascorbic acid(vitamin C), uric acid and acetaminophen.

As for oxidoreductase to be used in the method for quantitating asubstrate in accordance with the present invention, a suitable one canbe selected according to the type of substrate to dissolve in a samplesolution as an object to be measured. In a case where the substrate asan object to be measured is glucose, for example, oxidoreductase may beexemplified by glucose oxidase, pyrroloquinoline quinone-dependentglucose dehydrogenase, nicotinamide-adenine dinucleotide-dependentglucose dehydrogenase, and nicotinamide adenine dinucleotidephosphate-dependent glucose dehydrogenase; in a case where the substrateis cholesterol, oxidoreductase may be exemplified by cholesteroloxidase, nicotinamide-adenine dinucleotide-dependent cholesteroldehydrogenase, and nicotinamide adenine dinucleotide phosphate-dependentcholesterol dehydrogenase. Other than the aforesaid oxidoreductases, forexample, alcohol dehydrogenase, lactate oxidase, xanthine oxidase, aminoacid oxidase, ascorbic acid oxidase, acyl-CoA oxidase, uricase,glutamate dehydrogenase, fructose dehydrogenase, and the like, can beused according to the type of substrate as an object to be measured.

As examples of the electron mediator to be used in the method forquantitating a substrate in accordance with the present invention, metalcomplexes such as a ferrocene derivative, ferri/ferrocyanide ions,ruthenium hexacyanate, osmium-tris(bipyridynium) andosmium-di(bipyridynium)imidazolium, a quinone derivative such asp-benzoquinone, a phenazinium derivative such as phenazine methosulfate,a phenothiazinium derivative such as methylene blue, nicotinamideadenine dinucleotide, and nicotinamide adenine dinucleotide phosphatemay be cited.

An electron mediator having a high electron transfer rate against anenzyme to be used can be used favorably in combination with the enzyme.Among such electron mediators preferred are a ferrocene derivative,ruthenium hexacyanate, osmium-tris(bipyridynium) andosmium-di(bipyridynium)imidazolium, which are highly stable electronmediators.

Among them, electron mediators having a relatively high redox potentialare particularly preferred in implementation of the present invention inthat electric signals based respectively on a substrate and aninterfering substance, which are obtained in application of an ACpotential, can be obtained in a favorably separated manner.

These electron mediators may be in a linked form with a polymerbackbone, or in such a form that part or the whole thereof forms polymerchains. Further, oxygen can be used as the electron mediator. One typeor two of the above examples are used as the electron mediator.

In the following, the present invention is more specifically describedusing examples; however the present invention is not limited thereto.

EXAMPLE 1

10 mL of a phosphate buffer with pH 7, where 0.2 mM of ferrocenecarboxylic acid and 4 μM of glucose oxidase were dissolved as a reagentsystem, was obtained. This 10 mL phosphate buffer was put into acontainer made of Pyrex Glass to be used as an electrolyte, and acircular platinum disc with a diameter of 3 mm was used as a workingelectrode while a 2 cm-square platinum plate was used as a counterelectrode, to construct an electrochemical cell. A glucose aqueoussolution as a sample solution where ascorbic acid was dissolved wasadded such that the concentrations of ascorbic acid and glucose were 0.5mM and 20 mM (about 400 mg/dL), respectively.

Then, after the elapse of a certain period of time, an AC potential,having a central potential of +0.1 V relative to the counter electrodeand the amplitude of 0.01 V, was applied to the working electrode. Acentral potential at this working electrode was within the range of −0.1V to +0.1 V relative to a redox potential of ferrocene carboxylic acid.And the central potential was a potential more positive than a potentialthat was 0.05 V negative (0.13 V (pH 7) vs Ag/AgCl) relative to the mostnegative potential (0.18 V (pH 7) vs Ag/AgCl) in a potential regionwhere the reaction of ascorbic acid at the working electrode isdiffusion-controlled. The frequency of the AC potential was successivelyvaried from 16 mHz to 10 kHz, to be more precise, the value of (1.6,2.5, 4.0, 6.3 or 10)×(10⁻², 10⁻¹, 1, 10, 10² or 10³) Hz was used.

A certain period of time after the application of the AC potential,impedance Z was measured to plot complex impedance. As thus described,since there was almost no phase difference between the current andpotential at a relatively low frequency, the plot appeared in thevicinity of the real number axis; as the frequency was graduallyincreased, the magnitude of Z became smaller and the plot group formedan almost circular arc.

Next, series of the same electrolytes as thus described were prepared byvarying the glucose concentration. The ascorbic acid concentration wasfixed to be 0.5 mM and the respective glucose concentrations were 2, 3,4, 5 and 10 mM. Using these electrolytes, the measurement and plottingwere conducted in the same manner as above. As the electrolytes with thelower glucose concentrations were used, the diameter of the obtainedcircular arc gradually became longer, while having a certain correlationwith the glucose concentration. Such a behavior was also observed in acase where a glucose aqueous solution containing no ascorbic acid wasused as a sample solution.

It was therefore possible, by previously preparing an analyticalcalibration curve relating a diameter of a circle arc in an obtainedcomplex impedance plot with a glucose concentration, to determine theglucose aqueous solution concentration, without the influence ofascorbic acid, from a diameter of a circular arc of complex impedanceplot having been obtained in terms of a glucose aqueous solution with anunknown concentration. In this wise, the use of the method forquantitating a substrate in accordance with the present inventionallowed precise quantitation of a substrate contained in a samplesolution in a simply-structured measurement system without occurrence ofa measurement error due to an interfering substance.

EXAMPLE 2

In the present example, in place of the electrochemical cell used inExample 1, a biosensor produced in the following procedure was used toconduct measurement in the same manner as in Example 1. In the presentexample, a biosensor with a structure shown in FIGS. 1 and 2 wasproduced.

FIG. 1 is an exploded perspective view of the biosensor used in thepresent example, from which a reagent system has been removed. Aresin-made electrode pattern mask was placed on an glass-madeelectrically insulating base plate 1, and gold was sputtered to form aworking electrode 2 and a counter electrode 3. It should be noted that alayer comprising chrome was formed as an adhesive layer between the goldand glass so that the adhesive property therebetween was enhanced. Theworking electrode 2 and counter electrode 3 were electrically connectedto terminals for measurement outside the biosensor through means ofleads 4 and 5, respectively.

After formation of a layer of a reagent system comprising oxidoreductaseand an electron mediator on the working electrode 2, a spacer 7 having aslit 6 and a cover 9 having an air aperture 8 were bonded onto the baseplate 1 in such a positional relationship as shown by the broken linesin FIG. 1, to produce a biosensor. A sample solution supply pathway wasformed in the portion of the slit 6 in the spacer 7. The open end of theslit 6 at the end of the sensor was served as a sample supply openingfor the sample solution supply pathway.

FIG. 2 is a vertical sectional view of a biosensor in accordance withthe present invention. A reagent system 11 comprising oxidoreductase andan electron mediator was formed on the working electrode 2 formed on thebase plate 1. As shown in FIG. 2, the reagent system 11 was formed onthe electrode system comprising the working electrode 2 and counterelectrode 3.

When a sample solution was brought into contact with the open end of theslit 6 to serve as the sample solution supply pathway of the sensor withthe structure shown in FIG. 2, the sample solution was introduced intothe sample solution supply pathway due to capillary action to dissolvethe reagent system 11, and an enzyme reaction proceeded. Herein, as thesample solution supply pathway had previously been processed with anamphipathic reagent such as lecithin, the sample solution was introducedin a more uniform and smoother manner.

As thus described, when the base plate 1 where the electrode system wasplaced and the cover member, comprising the spacer 7 and the cover 9,were combined to form the sample solution supply pathway, between thebase plate 1 and the cover member, for introducing the sample solutionfrom the sample solution supply opening to the electrode system, theamount of the sample solution, containing the substrate as an object tobe measured, to be supplied to the sensor could be fixed so that theprecision of measurement could be improved.

In a sensor with the sample solution supply pathway provided therein,the reagent system might be disposed not only on the electrode system,but also in a portion exposed to the inside of the sample solutionsupply pathway in order that the reagent system would dissolve in asample solution to be supplied. The reagent system might for example beprovided in the portion of the cover 9 which was exposed to the insideof the sample solution supply pathway, and the portion not in contactwith the electrode system on the base plate 1 but exposed to the insideof the sample solution supply pathway. Moreover, the reagent systemmight be divided into a plurality of portions, one of which might beprovided on the base plate while the other be on the side of the covermember. In this regard, each of the divided layers was not necessarilyrequired to contain all the reagents. For example, oxidoreductase andthe electron mediator might be contained in separate layers.

Moreover, the insulating second base plate having uniting either thecounter electrode 3 or the working electrode 4 with either the lead 5 orthe lead 4 corresponding to the respective electrodes, might be used inplace of the cover 9. Also in this case, since the base plate 1, thespacer 7 and the second base plate formed the sample solution supplypathway, the amount of the sample solution to be supplied to the sensorcould be fixed so as to improve the precision of measurement.

The reagent system 11 was obtained by the use of ferrocene carboxylicacid and glucose oxidase, and 20 mM of a glucose aqueous solution as asample solution, where a 0.5 mM ascorbic acid was dissolved, was droppedinto the opening of the sample solution supply pathway of the sensor asthus produced, namely the open end of the slit 6 in the spacer 7, to besupplied to the sensor. After the lapse of a certain period of time, anAC potential, having a central potential of +0.1 V relative to thecounter electrode 3 and the amplitude of 0.01 V, was applied to theworking electrode 2. The AC potential had an equivalent frequency tothat described in Example 1. After the lapse of another certain periodof time, impedance Z was measured and complex impedance was plotted. Asa result, as in Example 1, the plot appeared in the vicinity of the realnumber axis at a relatively low frequency; as the frequency wasgradually increased, the magnitude of Z became smaller and the plotgroup formed an almost circular arc.

Next, as in the electrolyte described in Example 1, series ofelectrolytes with different glucose concentrations were prepared, andthese electrolytes were separately supplied to the sensor to conductmeasurement and plotting in the same manner as above. As theelectrolytes with lower glucose concentrations were used, the diameterof the obtained circular arc gradually became longer, while having acertain correlation with the glucose concentration. Such a behavior wasalso observed in a case where a glucose aqueous solution containing noascorbic acid was used as a sample solution.

It was therefore possible, by the same method as in Example 1, todetermine the glucose aqueous solution concentration from the diameterof the circular arc of the obtained complex impedance plot, without theinfluence of ascorbic acid. It was also possible to estimate the glucoseconcentration by conducting the aforesaid alternating currentmeasurement at a fixed single frequency and then using the magnitude ofobtained Z, “|Z|”. In this wise, the use of the method for quantitatinga substrate in accordance with the present invention allowed precisequantitation of a substrate contained in a sample solution in a simplystructured measurement system without occurrence of a measurement errordue to an interfering substance.

EXAMPLE 3

In the present example, an electrolyte (glucose concentration: 20 mM,ascorbic acid concentration: 0.5 mM) with the same composition as inExample 1 and an electrochemical cell were used. After the lapse of acertain period of time, an AC potential, which had a fixed singlefrequency, a central potential of +0.1 V when taking the counterelectrode as a reference and the amplitude of 0.01 V, was applied to theworking electrode. After the lapse of a certain period of time,impedance Z was measured. Next, a DC potential of +0.3 V relative to thecounter electrode was applied to the working electrode for a certainperiod of time to measure a direct current I′ flowing between theworking electrode and counter electrode.

As in Example 1, series of electrolytes with different glucoseconcentrations were used to conduct measurement and plotting in the samemanner as above. As the electrolytes with lower glucose concentrationswere used, obtained Z gradually became larger, having a certaincorrelation with the glucose concentration, as resulted in Example 1,and it was therefore possible to estimate the glucose concentration withthe use of the Z value.

Separately from the above case, a DC potential of +0.3 V relative to thecounter electrode was applied, or applied to the working electrode for acertain period of time, and then examined the relationship (I-G) betweenthe direct current I and the glucose concentration under non-existenceof ascorbic acid, as well as the relationship (I-A) between the directcurrent I and ascorbic acid under non-existence of glucose, by a methodfor measuring a direct current flowing between the working electrode andcounter electrode. It was then revealed that each of I-G and I-Aexhibited an almost favorable proportional relationship. On the otherhand, it was found that each of the relationship between the directcurrent I and the glucose concentration under the existence of a fixedconcentration of ascorbic acid, and the relationship between the directcurrent I and the ascorbic acid concentration under the existence of afixed concentration of glucose exhibited an almost favorable linerrelationship. It could thus be concluded that the direct current I′obtained by the aforesaid measurement was the sum of the respectivecurrents brought about by glucose and ascorbic acid.

As thus described, it was possible to estimate the glucose concentrationwith the use of the Z value obtained by the alternating currentmeasurement. When the glucose concentration as thus determined wasapplied to I-G, a current attributed to glucose among I′ obtained by theaforesaid measurement could be found, and by deduction thereof, acurrent attributed to ascorbic acid among I′ could be found. Applicationof this current to I-A could result in estimation of the ascorbic acidconcentration.

As thus described, according to the method for quantitating a substratein accordance with the present invention, quantitation of an interferingsubstance as well as measurement of a substrate could be conductedwithout the influence of the interfering substance. Respectiveanalytical curves in respect to glucose and ascorbic acid based on adirect current, namely I-G and I-A, might not be prepared for everymeasurement, but be obtained previously.

It should be noted that in the present example, after the step ofapplying an AC potential to measure Z, the step of applying a DCpotential to measure I′ was implemented; however, the order ofimplementing these steps might be reversed. Further, the samemeasurement could be conducted by means of a biosensor, as in Example 2.

EXAMPLE 4

In the present example, an electrochemical cell further comprising areference electrode in an electrode system was used. The sameelectrolyte, working electrode and counter electrode were used as thosein Example 1 and a silver/silver chloride electrode (Ag/AgCl electrode)was used as the reference electrode, to construct an electrochemicalcell. A glucose aqueous solution as a sample solution where ascorbicacid was dissolved was applied in such a manner that the concentrationsof ascorbic acid and glucose were 0.5 mM and 20 mM (about 400 mg/dL),respectively.

After the lapse of a certain period of time, an AC potential, which hada central potential of +0.36 V when taking the reference electrode as areference and the amplitude of 0.01 V, was applied to the workingelectrode. A central potential at this working electrode was within therange of −0.1 V to +0.1 V relative to a redox potential of ferrocenecarboxylic acid, and was a potential more positive than a potential thatwas 0.05 V negative (0.13 V (pH 7) vs Ag/AgCl) relative to the mostnegative potential (0.18 V (pH 7) vs Ag/AgCl) in a potential regionwhere the reaction of ascorbic acid at the working electrode wasdiffusion-controlled. The same frequency of the AC potential as that inExample 1 was used.

After the lapse of another certain period of time, impedance Z wasmeasured to plot complex impedance, and as in Example 1, the plotappeared on the real number axis at a relatively low frequency; as thefrequency was gradually increased, the magnitude of Z became smaller andthe plot group formed an almost circular arc.

Next, like the electrolytes described in Example 1, series ofelectrolytes with different glucose concentrations were prepared andthese electrolytes were used to conduct measurement and plotting in thesame manner as above. As the electrolytes with lower glucoseconcentrations were used, the diameter of the obtained circular arcgradually became longer, having a certain correlation with the glucoseconcentration. Such a behavior was also observed in a case where aglucose aqueous solution containing no ascorbic acid was used as asample solution.

It was therefore possible, by the same method as in Example 1, todetermine the glucose aqueous solution concentration from the diameterof the circular arc of the obtained complex impedance plot, without theinfluence of ascorbic acid. It was also possible to estimate the glucoseconcentration by conducting the aforesaid alternating currentmeasurement at a fixed single frequency and then using the obtainedmagnitude of Z, |Z|.

Moreover, since the use of the reference electrode allowed more stabletransfer of the potential of the working electrode, the obtained Z valuewas more stable compared with the case of Examples 1 and 2. In thiswise, the method for quantitating a substrate in accordance with thepresent invention enabled the substrate measurement to be more stablyimplemented, without the influence of an interfering substance.

Furthermore, a sensor was produced in the same manner as in Example 2,and immediately after the supply of the sample solution containingascorbic aid and glucose into the sensor, a silver/silver chlorideelectrode was brought into contact with the sample solution in thevicinity of the sample supply opening via a salt bridge comprisingpotassium chloride and agar. Except that an AC potential applied to theworking electrode had a central potential of 0.36 V relative to thereference electrode and the amplitude of 0.01 V, an alternating currentmeasurement was conducted in the same manner as in Example 2.Consequently, almost the same results as the results described inExample 2 were obtained. However, the obtained Z value was more stable.Hence, even in the case of using a reference electrode simultaneouslywith a sensor, according to the method for quantitating a substrate inaccordance with the present invention, it was possible to conduct thesubstrate measurement in a stable manner without the influence of aninterfering substance.

It should be noted that in the present example, although thesilver/silver chloride electrode was brought into contact with thesample solution in the vicinity of the sample supply opening via thesalt bridge, the similar effect can be obtained when the silver/silverchloride electrode was formed on the base plate of the sensor byscreen-printing and then used.

EXAMPLE 5

In the present example, first, 10 mL of a phosphate buffer with pH 7,where 1 mM of ruthenium hexacyanate and 0.6 kU/mL of pyloroquinolinequinone-dependent glucose dehydrogenase were dissolved as a reagentsystem, was obtained. This 10 mL phosphate buffer was put into acontainer made of Pyrex Glass to be used as an electrolyte, and acircular platinum disc with a diameter of 3 mm as a working electrode, a2 cm-square platinum plate as a counter electrode, and an Ag/AgCl(sat.KCl) as a reference electrode were used to construct anelectrochemical cell.

Subsequently, after the lapse of a certain period of time, an ACpotential, having a central potential of +0.9 V relative to thereference electrode and the amplitude of 0.01 V, was applied to theworking electrode. A central potential at this working electrode waswithin the range of −0.4 V to +0.4 V relative to a redox potential ofruthenium hexacyanate, and was a potential more positive than apotential that was 0.05 V negative (0.13 V (pH 7) vs Ag/AgCl) relativeto the most negative potential (0.18 V (pH 7) vs Ag/AgCl) in a potentialregion where the reaction of ascorbic acid at the working electrode wasdiffusion-controlled. The frequency of the AC potential was successivelyvaried from 16 mHz to 10 kHz, and more specifically, the same frequencyas that shown in Example 1 was used.

A certain period of time after the application of the AC potential,impedance Z was measured to plot complex impedance. As shown with ● inFIG. 3, in the absence of glucose, a plot forming almost a straight linewas obtained. When a glucose aqueous solution as a sample solution wasadded such that the concentration thereof was 10 mM, as shown with ▴ inFIG. 3, the plot group appeared in the vicinity of the real number axis;as the frequency was gradually increased, the magnitude of Z becamesmaller and the plot group formed an almost circular arc.

Next, ascorbic acid was added into the aforesaid electrolyte such thatthe concentration thereof was 0.5 mM. Measurement and plotting wereconducted in the same manner as above, to obtain the plot group showingalmost the same half circle as in the case of adding 10 mM of theglucose aqueous solution, as shown with ▪ in FIG. 3.

As thus described, even when a central potential of the AC potential tobe applied to the working electrode was within the range of −0.4 V to+0.4 V relative to a redox potential of an electron mediator, and was apotential more positive than a potential that was 0.05 V negativerelative to the most negative potential in a potential region where thereaction of the interfering substance at the working electrode wasdiffusion-controlled, the concentration of the glucose aqueous solutioncould be obtained from the diameter of the circular arc of the compleximpedance plot group. Further, even when an AC potential having acentral potential of 0.8 V, which was closer to the redox potential ofruthenium hexacyanate, was applied, the similar quantitation could beconducted. Moreover proved in the present example was that rutheniumhexacyanate functioned as a highly effective electron mediator in thepresent invention, and that pyloroquinoline quinone-dependent glucosedehydrogenase was a highly effective oxidoreductase in the presentinvention.

EXAMPLE 6

In the present example, the same electrochemical cell as in Example 1was used except that a platinum-made rotating disc electrode (RDE) wasused as a working electrode, and the same sample solution andmeasurement conditions as in Example 1 were employed for measurement.

As a result, substantially, almost the same results as those in Example1 were obtained; however, compared with the results of Example 1, thecircular arc diameter was shorter, and a plot was obtained on the realnumber axis at a higher frequency. This was because the use of the RDEcaused an increase in current variation derived from the electronmediator versus modulation of the potential applied to the workingelectrode, compared with the case of using a stationary electrode.

Next, like the electrolytes described in Example 1, series ofelectrolytes with different glucose concentrations were prepared, andused to conduct measurement and plotting in the same manner as above. Asthe electrolytes with the lower glucose concentrations were used, thediameter of the obtained circular arc gradually became longer, having acertain correlation with the glucose concentration. Such a behavior wasalso observed in a case where a glucose aqueous solution containing noascorbic acid was used as a sample solution. Even with the RDE usedinstead of a stationary electrode, almost no influence on the amount ofthe current variation derived from the interfering substance versus thepotential modulation was observed.

Accordingly, the glucose concentration could be measured with thediameter of this length. Worthy of note is that the variation in Z valueversus the variation in glucose concentration was larger than theresulted valuation in Example 1. It was also possible to estimate theglucose concentration by conducting the alternating current measurementat a fixed single frequency and using the obtained Z value. Such aneffect of using the RDE could also be obtained by using a stationaryelectrode and steadily stirring a solution which comprised a reagentsystem and a sample solution.

Further, almost the same result could be obtained in the case ofconducting the same measurement as in the present example by using, inplace of the RDE, a micro-electrode having an electrode area of such asize that a lateral diffusion of the electron mediator toward theelectrode surface was contributed to the electrochemical reaction.

A micro-electrode of a preferable size was one having a radius withinthe range of 20 μm to about 50 μm and an electrode area within the rangeof 1000 to 8000 μm² when the shape thereof was converted into a circle.As the electrode employed could be one produced by putting carbon fiber,platinum, gold or the like into a glass capillary to be sealed and thenmaking the surface of the electrode exposed, or a commercially availableone. Further, a micro-electrode produced on a base plate by utilizingsuch a semiconductor processing step as photo lithography or etchingcould also be used. The use of such a micro-electrode was favorableespecially when measurement was conducted using a biosensor as inExample 2.

As thus described, in the method for quantitating a substrate inaccordance with the present invention, the use of an RDE or amicro-electrode as the working electrode made stable and highlysensitive substrate measurement possible.

It should be noted that in the above examples, the central potential ofthe AC potential to be applied to the working electrode was +0.1 V whentaking the counter electrode as a reference, and +0.36 V or +0.9 V whentaking the reference electrode as a reference; however, the centralpotential is not limited to these values and may be in the vicinity of aredox potential of an electron mediator, and a favorable value can beselected according to the type of electron mediator to be used. Thisvalue also varies depending on whether the reference electrode is usedor not and a preferable value can be then selected.

Moreover, although the amplitude of the potential was 0.01 V in theabove examples, it is not limited to this value, and any amplitude withwhich the alternating current measurement can substantially be conductedmay be employed. While a value of usable amplitude is determined basedon performance of a measurement instrument, a preferable value is 1 to50 mV.

Although the frequency of the AC potential was 16 mHz to 10 kHz in theabove examples, it is not limited to these values and range. Anyfrequency at which an electric signal obtained by the alternatingcurrent method significantly varies according to a substrateconcentration may be employed.

Although the DC potential used in the above examples was +0.3 V whentaking the counter electrode as a reference, it is not limited to thisvalue; any potential with which an electron mediator and an interferingsubstance are sufficiently oxidized (or reduced) may be applied, and afavorable value can be selected according to the type of electronmediator to be used. Further, in the case of using the referenceelectrode, the DC potential can also vary depending on the type ofreference electrode and a favorable value corresponding thereto isselected.

Furthermore, the electric signal obtained by the application of the DCpotential was not limited to those described in the above examples. Itmay for example be a electrical charge passed at a working electrode.

The certain period of time for the reaction, which was used in the aboveexamples, may be a period of time for which output of an observabledegree can be obtained in almost a stable manner in implementation ofthe present invention.

Although platinum or gold was used as the electrode material in theabove examples, it is not limited thereto. As other examples ofelectrodes using different materials cited can be an electrodecomprising palladium and an electrode comprising carbon. Further, anelectrode using a mixed material primarily comprising any one ofplatinum, gold, palladium and carbon can also be employed. Although theelectrode comprising the working electrode and counter electrode, whichcomprised the equivalent material, was used in the above example, eachelectrode may be produced using different materials for the respectiveelectrodes.

Although the spattering method through a mask was used as the productionmethod of the electrodes in the biosensor as well as the productionmethod of patterns of the electrodes in the above examples, the methodswere not limited thereto, and for example, the pattern may be producedby combining a metal film formed by any of spattering, ion-plating,vapor deposition and chemical vapor deposition methods, with photolithography and etching. The pattern can also be formed by trimmingmetal by means of a laser. The electrode pattern may also be formed byconducting screen-printing on a base plate with the use of a metalpaste. Furthermore, patterned metal foil may be bonded onto a base plateas it is. When the electrode material is one mainly composed of carbon,the electrode pattern may be formed by conducting screen-printing on abase plate.

The shapes, dispositions, numbers, sizes and the like of these electrodesystems are not limited to those described in the above examples. Forexample, a working electrode and a counter electrode may be formed ondifferent insulating base plates, and a plurality of working electrodesand counter electrodes may be formed. The shape thereof may becomb-shaped. Further, the shapes, dispositions, number, sizes and thelike of leads and terminals are not limited to those described in theabove examples.

There is no limitation on the rotating rate of the RDE. Further, theelectrode area of the micro-electrode may be within the range of such asize that a lateral diffusion of the electron mediator toward theelectrode surface contributes to the electrochemical reaction.

The concentrations of the substrate and interfering substance in thesample solution are not particularly limited to those values describedin the above examples. The sample solution amount is also not limited.

The amount or the concentration of each reagent contained in the reagentsystem is not limited to the values described in the above examples. Itmay be an amount with which an enzyme reaction and an electrochemicalreaction proceed sufficiently.

An enzyme and an electron mediator may be in the insolubilized state orthe non-eluted state by immobilizing, on the working electrode, thewhole reagent system or one or more of reagents out of the reagentscontained in the reagent system in the above examples. In the case ofsuch immobilization, a covalent binding method, a cross-linking method,or a fixing method using interaction of coordinate bond and a specificaffinity is preferably used. A method of surrounding an enzyme and anelectron mediator by a polymer substance to give a pseudo-immobilizedstate is also effective as a method for readily forming a reagentsystem. A polymer to be used may be hydrophobic or hydrophilic, and thelater is more preferable. As examples of hydrophilic polymers cited canbe hydrophilic cellulose derivatives such as carboxy methyl cellulose,hydroxyethyl cellulose and ethyl cellulose, poly vinyl alcohol, gelatin,polyacrylic acid, starch and a derivative thereof, a maleic anhydridepolymer, and a methacrylate derivative.

Further, it is more preferable that a pH buffer is contained in thereagent system in the above examples. This enables adjustment of pH of areaction solution to be a suitable value for enzyme activity so that anenzyme can function effectively at the time of measurement. Moreover,since many interfering substances electrochemically react whileinvolving a proton, a potential region where the reaction of theinterfering substance is diffusion-controlled varies depending on pH ofan electrolyte, but the use of the pH buffer allows the potential regionto be fixed to certain values. It is therefore possible to stably removethe influence of the interfering substance in the present invention. Asfor the pH buffer used can be one containing one or more of phosphate,acetate, borate, citrate, phthalate and glycine. A buffer containing oneor more of hydrogen salts out of the above salts may also be used. Areagent to be used as so-called “Good's buffer” may also be used. Theforms of these pH buffers when contained within the sensor system canvary according to a sensor structure, and for example, they may be solidor solutions.

In the biosensors described in the above examples, it is preferable thata spacer is comprised as a constituent of the biosensor because theprecision of measurement can be improved by readily and uniformlydefining an amount of a solution containing a substrate as an object tobe measured. However, in the case of using the biosensor described inthe present invention in combination with an appliance capable of takinga fixed volume of a sample, a cover member comprising a spacer and acover is not necessarily required.

Industrial Applicability

As thus described, according to the present invention, there can beprovided a method capable of precisely quantitating a substratecontained in a sample solution by means of a measurement system with asimple structure, without occurrence of a measurement error due to aninterfering substance.

1. A method for quantitating a substrate in a sample solution, whichcontains a dissolved interfering substance and said substrate, by theuse of an electrode system and a reagent system, comprising the stepsof: (a) supplying a sample solution which contains a dissolvedinterfering substance and a substrate to an electrode system comprisinga working electrode and a counter electrode under the existence of areagent system comprising oxidoreductase and an electron mediator; (b)applying an AC potential to said working electrode to cause a redoxreaction of said electron mediator: (c) measuring an electric signalproduced on the basis of said redox reaction, by means of said electrodesystem; and (d) quantitating said substrate on the basis of saidelectric signal.
 2. The method for quantitating a substrate inaccordance with claim 1, characterized in that in said step (a), saidworking electrode and said counter electrode are disposed on the sameplane.
 3. The method for quantitating a substrate in accordance withclaim 1, characterized in that in said step (a), said working electrodeand said counter electrode are disposed in positions opposed to eachother across a space.
 4. The method for quantitating a substrate inaccordance with claim 1, further comprising a step (e) of applying a DCpotential to said working electrode, and a step (f) of measuring anelectric signal produced in said step (e).
 5. The method forquantitating a substrate in accordance with claim 1, characterized inthat in said step (b), a central potential of said AC potential iswithin the range of −0.4 to +0.4 V relative to a redox potential of saidelectron mediator, and is a potential more positive than a potentialthat is 0.05 V negative relative to the most negative potential in apotential region where the reaction of said interfering substance atsaid working electrode is diffusion-controlled.
 6. The method forquantitating a substrate in accordance with claim 1, characterized inthat in said step (b), a central potential of said AC potential iswithin the range of −0.1 to +0.1 V relative to a redox potential of saidelectron mediator, and is a potential more positive than a potentialthat is +0.05 V relative to the most negative potential in a potentialregion where the reaction of said interfering substance at said workingelectrode is diffusion-controlled.
 7. The method for quantitating asubstrate in accordance with claim 1, characterized in that saidelectric signal is impedance.
 8. The method for quantitating a substratein accordance with claim 1, characterized in that said electrode systemfurther comprises a reference electrode.
 9. The method for quantitatinga substrate in accordance with claim 1, characterized in that saidworking electrode is a rotating disc electrode or a micro-electrode. 10.The method for quantitating a substrate in accordance with claim 1,characterized in that said oxidoreductase is glucose oxidase orpyrroloquinoline quinone-dependent glucose dehydrogenase, and saidelectron mediator is ferrocene carboxylic acid.
 11. The method forquantitating a substrate in accordance with claim 1, characterized inthat said oxidoreductase is pyloroquinoline quinone-dependent glucosedehydrogenase, and said electron mediator is ruthenium hexacyanate.